1. Field of the Invention
This invention broadly relates to a new method of culturing freeze-dried microorganisms, as well as to a new method of freeze-drying microorganisms and the product resulting therefrom. The present invention particularly pertains to a method of freeze-drying microorganisms in a vessel with nutrients so as to produce a freeze-dried preparation containing viable microorganisms that undergo substantial logarithmic or exponential growth directly in the vessel upon rehydration with sterile, distilled water. This invention has utility in diagnostic applications, such as in research and in clinical laboratories, and as an educational tool.
2. Description of the Prior Art
Freeze-drying has long been considered a simple and economical way to preserve cultures for future experimentation, reference or comparison. Freeze-drying produces a dry preparation generally capable of being stored for long periods of time while retaining biological activity. The process is broadly described in American Type Culture Collection Methods, I. Laboratory Manual on Preservation: Freezing and Freeze-Drying, Hatt, H. (ed.), A.T.C.C. (1980) with specific reference to processes suitable for different microorganisms. The above-referenced manual describes both the conventional procedures suitable for freeze-drying a wide variety of microorganisms and equipment suitable for carrying out the procedures.
When the moisture content of a culture is removed during the freeze-drying process, molecules are virtually locked in position so that little or no opportunity exists for alteration of the physical or chemical properties of the product. Obviously, in preparing such products it is of prime importance that the viability of the culture be maintained. In order to protect the organisms, the freeze-drying process generally is carried out in the presence of a variety of cryoprotectant agents, designed to minimize cellular damage and increase survivability of microorganisms during the freeze-drying process. Cryoprotectant agents used in the prior art have included glucose, sucrose, lactose, mannitol, glycerol, dextran, fructose, and other materials such as monosodium glutamate, polyvinylpyrrolidone (PVP), sweet whey solids, dried skim milk, dried whole milk and bovine serum albumin.
It is also common to include a small amount of fresh nutrient media (e.g., nutrient broth) in the concentrated microorganism culture prior to freeze-drying. The nutrient media is used as a biologically compatible suspending agent. The nutrient media may also function as a cryoprotectant and also provides an immediate food source for the microorganisms upon their revitalization. The prior art, however, does not disclose a process for freeze-drying microorganisms in a vessel wherein the proportion of microorganisms to nutrient media in the suspension to be freeze-dried is adjusted so that when the freeze-dried preparation is properly rehydrated with sterile, distilled water, an active, growing culture is produced in situ.
The prior art always has used a concentration of organisms in relation to the quantity of nutrients added with the suspending liquid such that standard rehydration of the freeze-dried preparation with sterile, distilled water produces a non-dormant culture in a substantially stationary growth phase of development. Rehydration generally is accomplished by mixing the freeze-dried preparation with suitable nutrient broth, sterile distilled water, etc. Thereafter, propagation of the culture normally is accomplished by inoculating freshly prepared growth media with all or part of the rehydrated preparation. In other words, freeze-dried preparations of microorganisms always have been used to propagate numerous fresh cultures using freshly prepared, external nutrient medium rather than being used directly in the vessel in which they are stored to propagate a single culture in situ.
It is an object of the present invention to provide a simple method of establishing a growing culture in a vessel, such as in a small vial, from a freeze-dried preparation of microorganisms.
It is also an object of the present invention to provide a method of reconstituting a freeze-dried preparation of microorganisms which eliminates the need for preparing or supplying fresh nutrient medium with the freeze-dried preparation in order to develop an actively growing and multiplying culture from the freeze-dried preparation of microorganisms.
It is another object of this invention to provide a method of freeze-drying microorganisms and nutrients in a vessel so that an active, growing culture is produced in situ merely by rehydrating the freeze-dried preparation with sterile, distilled water.
It is still another object of this invention to provide a vessel having a specific freeze-dried composition of microorganisms, nutrients and optionally a cryoprotective agent, such that simple addition of a proper amount of sterile, distilled water to the vessel produces an active, growing culture therein.
These and other objects which will become apparent from the following description are provided by the present invention.